Summary of Work: The objective has been to test the hypothesis that there is/not a positive correlation between the presence of specific chemical contamination in aquatic environments and the incidence of abnormality among frogs from sites previously identified in Minnesota as normal vs abnormal. The focus of the investigation is on the effects of actual environmental mixtures rather than specific single compounds. The experimental design has consisted of comparative water chemistry on samples taken at timed intervals from previously identified sites based on field observations of malformed frogs. The water analysis is concommitant with scoring for incidence of abnormality among the field frogs at various developmental stages. Water samples, and fractions are also being tested for the capacity to malform frog embryos in the laboratory. Semipermeable membrane concentrators developed by the USGS have been deployed to mimic biological membrane transport and concentrate unknown compounds such as PAH's, retinoids and furans. Field studies have confirmed that there is a significant elevation in the frequency of malformed amphibians at several pond sites. Mixtures of factors that cause frog malformation in the laboratory have been isolated from affected sites. Some groundwater sources may be transporting teratogenic agents. It has also been determined that pathways associated with thyroid metabolism may be involved and that there are potentiating effects when certain water fractions are combined. Additional investigation will include gene markers for frog embryogenesis, retinoid/thyroid receptor binding, endocrine response and transgenic fish mutagensis assays in combination with specific mixtures from the affected sites. One important observation is that very complex mixtures of manmade chemical degradation products and natural compounds may be acting in the environment to produce effects that are different from results when chemicals are tested individually.